Lipoprotein lipase (LPL) is the rate-limiting enzyme controlling plasma triglyceride (TG) levels by hydrolyzing TG into free fatty acids and glycerol. This biological process is critical for energy mobilization and utilization and has a variety of clinical implications. LPL is specifically found in endothelial cells lining the capillaries.
Mutations that cause LPL deficiency result in type 1 hyperlipoproteinemia, hypertriglyceridemia or other disorders involving lipoprotein metabolism.
Traditionally, a variety of assays have been employed for measuring LPL activity. Generally, they can be divided into two formats, coupled and uncoupled assays. Coupled assays involve a chain of enzymatic reactions and can be used to indirectly determine LPL activity in relatively simple biological specimens. Uncoupled assays can further be divided to two groups, non-homogeneous and homogeneous assays.
Non-homogeneous assays require quantifying lipolytic products, typically by monitoring fatty acids liberated by LPL. Non-homogeneous assays are tedious and have a low throughput. For example, a radiolabeled triolein substrate is typically used in radiometric assays for measuring LPL activity. The labeled fatty acids released by LPL must be separated from the labeled triolein, before quantification, through a series of organic extraction procedures. Another common non-homogeneous assay used is a titratimetric method, which is not very sensitive.
Homogeneous assays are typically fluorescence-based and rely on changes in the fluorescence properties of a substrate upon hydrolysis. Traditionally, several naturally fluorescent groups including Dansyl, NBD or Pyrene have been incorporated into TG substrates for detecting LPL activity. However, the high background noise created by using these groups limits their wide use. Coumarin derivatives are one fluorescent group which only fluoresce after they are converted to lipolytic products, but these derivatives suffer from many problems such as instability and non-specific hydrolysis by enzymes other than LPL. Further, they do not resemble LPL's native substrate, triglyceride.
What is desired is an easy to use, sensitive, environmentally friendly, real-time LPL assay which does not require purification and will provide accurate results at a high throughput.